Polypeptide compounds and nucleotide sequences promoting resistance to eutypa dieback in plants

ABSTRACT

Polypeptide compounds and nucleotide sequences promoting resistance to eutypa dieback in plants The subject of the invention is a nucleotide sequence coding for an enzyme with eutypine reductase activity, capable of metabolizing the eutypine synthesized in plants by a fungus of the Eutypa lata or Libertella blepharis type. The overproduction of eutypine reductase by the plant host of the fungus enables the consequences of the presence of this fungus in plants to be attenuated or even eradicated.

BACKGROUND OF THE INVENTION

The subject of the invention is compounds implicated in resistance toeutypa dieback in plants as well as agents for combatting this disease,for example in the grapevine.

At present eutypa dieback is the most serious disease of the grapevine.In fact, it is present in vineyards throughout the world; it leads tothe premature death of many grapevines of the sensitive varieties andthere exist no curative agents. The parasite responsible for eutypadieback of the grapevine is an ascomycete fungus existing in a perfectform Eutypa lata (Carter M. V., 1955, Apricot gummosis--a newdevelopment J. Dep. Agric. South Aust. 59: 178-184) Tul and C.Tul. Syn;E armeniacae Hansf. and Carter) or an imperfect form Libertellablepharis (Samuel G, 1933 "Gummosis" or "Dieback" in apricot trees J.Dep. Agric. South Aust. 36: 979-980) A. L. Smith (syn. Cytosporina sp.).

Eutypa dieback is certainly an ancient disease, even though thepathogenic agent was observed on the grapevine only in 1973 inAustralia. In France the disease was identified with certainty by Bolayin 1977 in the region of Languedoc-Roussillon. From 1978, theobservations have multiplied in the vineyards of France which show theexpansion of eutypa dieback. The reasons for the spread of this diebackdisease, which is endemic, have still not been dearly identified to-day;according to certain authors, this progression might be attributable tothe modification of certain cultural practices.

The grapevine is not the only perennial ligneous plant which is host tothis parasite. In fact, the signs of eutypa dieback have been observedin particular on trees and bushes such as the apricot, black currant,cherry, tamarind, almond, apple . . . .

The disease affects the trunk and the "arms" of the grapevine, sites ofdevelopment of the mycelium, and the herbaceous parts which, however,never harbour the parasite. The ascospores of the fungus, released bythe asci of the perithecia situated on the rind of the contaminatedgrapevines, penetrate via the wounds resulting from pruning into thelumen of the vessels of the xylem, then germinate at several millimetersfrom the surface of the wound and colonize the xylem, the cambium andthe phloem. The installation of the mycelium in the "wood" of the trunkand the arms of the grapevine leads to the formation of an alwayswell-defined, hard and sectorial, brown necrosis, which has anappearance of dry rot, a sign of a preferential degradation of thecellulose of the wood.

The plants affected by eutypa dieback show typical symptoms in thespring. In the most characteristic cases, the branches are emaciatedwith short internodes; the leaves of small size, often coiled or evendeformed, have a slightly chlorotic appearance and sometimes smallmarginal necroses. The inflorescences seem to develop normally untilflowering, but they may then necrose or give bunches of small grapes(fruit drop). The symptoms described often affect one arm but thedisease can then spread to all of the grapevine, leading to the death ofthe latter.

The duration of the slow incubation of the fungus in the trunk and thearms (3 to 10 years) confers on this disease an insidious character. Theexpression of the symptoms on the herbaceous organs is uncertaindepending on the year: a contaminated grapevine does not show symptomsregularly, certainly for reasons related to environmental factors and inparticular to climatic conditions. Moreover, the existence of isolatesof different aggressivity has been demonstrated, making still moredifficult the recognition of the disease.

The destruction of the parasite in the trunk of the grapevines or thereduction of its expression is not possible to-day. In the absence ofcurative pest control measures, different prophylactic and preventivemeasures have been recommended to contain the disease. It isrecommended, for example, to limit the pressure of the inoculum byremoving old grapevines and the arms cut after the pruning operation.Awareness of the conditions of release and germination of the ascosporeshas led to several recommendations: prune late while taking account ofthe climatic conditions (dry and calm weather), avoid prunings causinglarge surface wounds, protect each pruning wound by the manualapplication of a fungicide to limit the germination of the ascospores.Finally, the affected strains may be restored by the operation ofcutting them back severely, which consists of preserving a stem existingbelow the diseased part of the trunk and of removing the upper part.

It can be seen that eutypa dieback is certainly harmful because thepresent pest control agents, uniquely prophylactic and preventive, arerestrictive, expensive and of limited scope.

The economic impact of this disease is considerable. The surveysconducted in France since 1988 show that the levels of symptoms arevariable and depend on the variety of grapevine and, for a givengrapevine, depend on the year and the regions, sometimes up to 50% ofthe grapevines being subjected to the disease. The most sensitivevarieties include the Sauvignon blanc, the Cabernet Sauvignon, the Ugniblanc, the Cinsault and the Chein whereas the Merlot and the Semillonare the most tolerant.

A network of observations based on a rigorous statistical approach setup in the context of a European contract ("Eutypa dieback control"programme No. 8001-CT-91.205) focussed on 85000 grapevines cultivated in11 wine-growing regions and on 10 varieties of grapevine. It made itpossible to show that all the varieties exhibit symptoms of eutpadieback but to very variable extents: 30 to 80% in certain regions ofGreece, 5 to 10% in the Rioja Alta region (Spain, Tempranillo variety)and in Italy (Trebbiano variety) and less than 2%, even 1%, in thewine-growing areas of Portugal.

This disease may also entail a reduction of the expression of thearomatic quality in certain grapevine varieties. In addition, eutypadieback requires the premature replacement of the dead varieties whichcauses the rejuvenation of the vineyard, which is detrimental toquality. It should also be noted that the parasitic fungus plays apioneer role in the installation of parasitic apoplexy (Carignon C.,1991), fungal complex also responsible for a dieback of the grapevine.

This disease also has indirect effects on quality by modifying theplanting of the vines in the vineyards because the sensitive varietiesare little by little replaced by more tolerant varieties, while howeverrespecting the restrictions of the registered designation of origin.Furthermore, if the harvest volume is maintained in spite of varietiesmissing to a high extent, the quality of the wines may be affected.

Consequently, it is dear that for the winegrowers wishing to maintain aregular production of quality wines, eutypa dieback is presently themost worrying disease.

The inventors have shown that eutypa dieback is associated with thesynthesis of a compound of the family of the lipophilic acids,4-hydroxy-3-(3-methylbut-3-en-1-ynyl) benzaldehyde, called eutypine(Tey-Rulh P. et al. Phytochemistry, vol. 30, No. 2, pp. 471-473, 1991)in some tissues of the host harbouring the parasitic fungus. This toxicmolecule is synthesized by the parasitic fungus.

In order to control this disease the inventors have been concerned withthe structure and physiology of eutypine when it is synthesized in thehost by the parasite and have observed in plants and in particular inthe plants resistant to eutypa dieback, the synthesis of degradationproducts of this toxic constituent.

By the expression "plants resistant to eutypa dieback" is meant in thecontext of the present application, the capacity of the plantscontaminated by the parasite responsible to resist the harmful effectsinduced directly or indirectly by the presence of the parasite.

This type of resistance may also be explained as the capacity of theplants contaminated by the fungus responsible for eutypa dieback totolerate the presence of this parasite without their development,including their growth and their physiology, being affected to an extentwhich would entail the dieback of the plants in question.

The inventors have demonstrated a metabolic pathway for eutypine withinthe plants contaminated by the parasite, resulting in the synthesis ofproducts non-toxic for these plants; as a result, they have suggestedagents for conferring on plants sensitive to eutypa dieback propertiesof resistance or, where appropriate, agents for increasing theresistance capacities already existing in certain plants towards eutypadieback.

In this connection, the inventors have identified and characterized inparticular a polypeptide with enzymatic activity of the reductase typecapable of promoting the degradation of the toxic eutypine intonon-toxic constituents including a compound called eutypinol.

The inventors have isolated and identified the eutypine reductase enzymeand have succeeded in characterizing the nucleotide sequence coding forthis enzyme.

The nucleotide sequence coding for this enzyme thus constitutes a novelmeans for considering the production of plants resistant to eutypadieback in conformity with the definition which was given previously ofthis resistance. For example, the characterization of this nucleotidesequence makes it possible to prepare transgenic plants capable ofoverproducing eutypine reductase and of increasing the metabolism of theeutypine produced in the host by the parasite.

SUMMARY OF THE INVENTION

Hence the subject of the invention is a nucleotide sequence,characterized in that it codes for a polypeptide with eutypine reductaseactivity, this polypeptide comprising the amino acid sequencerepresented in FIG. 1 (SEQ ID NO.3).

According to a particular embodiment of the invention, this nucleotidesequence is characterized in that it comprises the nudeotide sequenceincluded between the nucleotides 22 and 975 of the nucleotide sequenceshown in FIG. 1 (SEQ ID NO.3).

As a variant, such a sequence is characterized in that it corresponds tothe sequence shown in FIG. 1 (SEQ ID NO.3), which is a complementary DNAsequence (cDNA) of the VRER gene coding for the eutypine reductasemolecule.

The subject of the present invention is also a nucleotide sequencehybridizing with one of the sequences previously described under highlystringent conditions.

Such highly stringent conditions are the following: placing in contactwith a solution containing SSC (2×), 1% SDS for 5 min at 25° C., thentwice for 15 min. at 45° C., then in a solution containing SSC (0.2 ×),1% SDS twice for 15 min. at 45° C.

Alternatively, the nudeotide sequence of the invention may be defined inthat it possesses a sequence homology of more than 75% with thenucleotide sequence included between the nucleotides 22 and 975 of thesequence shown in FIG. 1 (SEQ ID NO.3), or with the nudeotide sequenceshown in FIG. 1 (SEQ ID NO.3).

Preferably, this homology is greater than or equal to 80% andadvantageously greater than or equal to 90%.

The nucleotide sequences may advantageously be used in the procedures ofgenetic engineering to produce the compounds according to the invention.

In this connection, a sequence should advantageously be used whichcomplies with one of the preceding definitions under the control of ahomologous or heterologous promoter, and preferably under the control ofa promoter suitable for expression in prokaryotic and/or eukaryoticcells.

Preferably, and depending on the level of eutypine reductase activitydesired, recourse should be had to a strong promoter for expressing anucleotide sequence according to the invention. A suitable promoter isfor example the strong promoter 35S of the CaMV (EP-B-0131623)permitting expression in plant cells.

Where appropriate, the promoter used is combined with other regulatorysequences such as expression enhancers.

As examples of vectors, mention should be made of the binary vector pGAdescribed by An, G et al., 1988, Binary Vectors, pp 1-19 in PlantMolecular Biology Manual A3 Kluwer Acad. Pub., Dordrecht, TheNetherlands.

The application also relates to recombinant prokaryotic or eukaryoticcells, containing a nudeotide sequence complying with one of thedefinitions given above.

Valuable recombinant cells in the framework of the embodiment of theinvention are for example bacterial cells, in particular E. coli orAgrobacterium tumefaciens cells. They may also be eukaryotic cells and,in particular, plant cells. As examples, recourse should be had to cellsof plant seeds or to grapevine cells, in particular when it is desiredto implement resistance to eutypa dieback in the grapevine.

Also included in the framework of the invention are a biological tissueor plant producing a polypeptide compound complying with one of thepreceding definitions after transformation by a nucleotide sequenceaccording to the invention. Preferably, the recombinant plant is thegrapevine.

The invention also relates to seeds obtained from said plants as well asto a process for the production of these plants or these seeds.

Preferably, the cells, tissues and plants transformed in the context ofthe invention contain the nudeotide sequence integrated stably intotheir genome. Any suitable transformation procedure may be used, such aselectroporation, bombardment with microprojectiles carrying DNA, forexample, by means of a particle gun, the co-culture of explants in thepresence of Agrobacterium tumefaciens.

Such a production process for a plant or a seed expressing a polypeptidecompound with eutypine reductase activity is characterized in that itcomprises the steps of

a) transformation of a plant cell with a nucleotide sequence definedabove or a vector defined above under conditions permitting the stableand functional expression of the protein with eutypine reductaseactivity encoded in the above-mentioned nucleotide sequence;

b) regeneration of plants from the transformed plant cell of step a) toobtain plants expressing the protein with eutypine reductase activity;

c) where appropriate, production of seeds from the modified plantsobtained in step b).

The process of the invention may advantageously be implemented toproduce plants of the grapevine family, but also to produce plantsresistant to eutypa dieback when these plants are naturally sensitive tothis disease, for example perennial ligneous plants such as the apricot,black currant, cherry, tamarind, almond or apple.

Moreover, the subject of the invention is the use of the nucleotidesequence such as defined in the preceding pages as selection agent, adetector of the transformation of cells by means of a defined nucleotidesequence whose expression is coupled to that of the sequence used asselection agent. In the context of this application the sequence or partof the sequence of the VRER gene or its cDNA may be used as selectiongene for making it possible to identify cells, tissues or plantsgenetically transformed by a sequence of interest. In this case, theselection gene is transferred with the gene of interest to the cellsthat it is desired to transform. The sequence of the gene of interestand that of the selection gene are combined so that the integrationand/or the expression of the one is linked to the integration and/or theexpression of the other. The value of using the sequence of theinvention as selection gene or sequence lies in its making it possiblein particular to avoid certain problems linked to the dissemination ofthe genes, a problem encountered with the genes for resistance toantibiotics or herbicides. In the framework of this application, thetransformed cells, tissues or plants may be selected by being placed incontact with eutypine or with any substrate capable of being reduced bythe eutypine reductase produced by the selection gene.

The selection of the transformed cells, tissues or organisms which haveintegrated the selection gene with the aid of eutypine or any suitablesubstrate should be feasible by prior determination of the thresholdsensitivity to eutypine of the cell of the tissue or plant considered.This determination should be made by procedures similar to those knownfor the determination of the sensitivity of a given species or organismto an antibiotic or herbicide.

Hence the subject of the invention is also a recombinant nucleic acidcontaining a nucleotide sequence according to the invention, used asselection agent in combination with a sequence of interest, saidcombination making possible the linked integration of the selectionmarker and the sequence of interest in a defined cell host.

Consequently, the framework of the invention includes a procedure forthe detection of cells transformed by a defined sequence of interestcomprising:

a) the transformation of the cells with a recombinant nucleic acidcomprising the gene of interest and a selection gene comprising anucleotide sequence coding for a polypeptide with eutypine reductaseactivity according to the invention,

b) the placing of the cells obtained in step a) in contact with asubstrate for eutypine reductase,

c) the detection of a reduction reaction undergone by the substrate

d) where appropriate, the selection of the transformed cells giving riseto this reduction reaction.

Hence the subject of the invention is also a polypeptide compound witheutypine reductase activity, characterized in that it comprises theamino acid sequence described in FIG. 1 (SEQ ID NO.3).

According to this definition, the eutypine reductase activity observedis the capacity conferred on the host of the parasite responsible foreutypa dieback or increased by this polypeptide compound to degrade ormetabolize eutypine to degradation products non-toxic for the referencehost usually sensitive to the toxicity linked to the presence ofeutypine.

Eutypine or 4-hydroxy-3-(3-methylbut-3-en-1-ynyl) benzaldehyde is alipophilic weak acid which has been demonstrated to be capable of beingdegraded to a hydroxylated derivative,4-hydroxy-3-(3-methylbut-3-en-1-ynyl) benzylic alcohol, called eutypinolin the context of the present application, as a result of an enzymaticreaction for which the constituent NADPH is a specific co-factor whenthe reaction is performed in vitro.

In an environment permitting it, the polypeptide compound according tothe invention is hence capable of carrying out this enzymatic reactionto metabolize eutypine to a non-toxic derivative, in particular to theform of eutypinol.

Where appropriate, the eutypine reductase activity may be observed onother aldehydic substrates.

The polypeptide compound previously described is characterized by thepresence in its structure of an amino acid sequence, the sequence ofwhich has been given in FIG. 1 (SEQ ID NO.3); if necessary, it mayinclude additional groups the presence and/or nature of which dependsfor example on the cell host in which it is expressed.

A valuable compound according to the invention is also characterized inthat it has a molecular weight of about 36 kDa, calculated from thecoding sequence and measured on the purified protein.

According to a particular embodiment of the invention, the polypeptidecompound is characterized in that it is encoded in a nucleotide sequencecomprising the coding nucleotide sequence included between thenucleotides 22 and 175 (these end nucleotides being included) of thesequence shown in FIG. 1 (SEQ ID NO.3).

The subject of the invention also includes compounds modified withrespect to the polypeptide compound previously defined. In particular,they may be polypeptide compounds comprising an amino acid sequencederived from the amino acid sequence described in FIG. 1 (SEQ ID NO.3),for example by addition, deletion or substitution of at least one aminoacid residue contained in this sequence, the compound formed having aeutypine reductase activity according to the definition which is givenabove.

When the compound of the invention is obtained by deletion of at leastone amino acid residue of the sequence of the polypeptide compoundpreviously described, the deletion may extend to the elimination of oneor more fragments unnecessary or inessential for the eutypine reductaseactivity of the compound complying with the definition of FIG. 1 (SEQ IDNO.3).

Similarly, when a derivative of the compounds described above isobtained by substitution of certain amino acid residues, they may bereplaced by neutral residues or by residues having the same propertiesor by residues capable of promoting the desired reducing properties.

According to a variant of the embodiment of the invention, thepolypeptide compound is characterized in that it is recognized byantibodies obtained against a polypeptide complying with the amino acidsequence shown in FIG. 1 (SEQ ID NO.3).

Such antibodies are for example polyclonal antibodies obtained byimmunization of an animal, in particular a rabbit, with the polypeptidecompound with eutypine reductase activity according to the invention andrecovery of the antibodies formed from the rabbit serum.

Also included in the context of the invention is a polypeptide compoundcharacterized in that its amino acid sequence exhibits a sequencehomology of at least 50% with the amino acid sequence shown in FIG. 1(SEQ ID NO.3).

By homology is meant the identity of the amino acids compared when thetwo amino acid sequences are compared or, in the absence of identity,the conservation of the properties of the original amino acid in thesubstituted amino acid.

Advantageously, the homology previously in question is equal to at least70%, and preferably equal to at least 80, even 90%, in numbers of aminoacid residues, this homology being evaluated by reference to the aminoacid sequence shown in FIG. 1 (SEQ ID NO.3).

The polypeptide compounds according to the invention may be obtained byany suitable method and in particular may be isolated from plants, forexample from the bean Vigna radiata (Mung Bean) or may also besynthesized chemically or prepared by genetic engineering in particularin recombinant cells.

Other plants may also be used to extract the compounds of the invention,such as the grapevine, the potato, the carrot, etc . . .

Other characteristics and advantages will become apparent in the Figuresdescribed below, as well as in the examples.

BRIEF DESCRIPTTION OF THE DRAWINGS

FIG. 1: Nucleotide sequence of the full length cDNA of the VRER gene andpeptide sequence deduced.

FIG. 2: Schematic representation of the binary vector pGA-ER. RB and LB:right (700 bp) and left (600 bp) borders of T-DNA; OriV: origin ofreplication (E. coli and Agrobacterium); OriT: origin of conjugativetransfer; trfA: "trans" replication factor; tet^(R) : gene forresistance to tetracycline; NPTII: neomycin phosphotransferase gene(resistance to kanamycin) under the dependence of the nos (nopalinesynthase) promoter and terminator; 35S: strong promoter of CaMV; 5' and7': transcription terminators of the genes 5 and 7 of pTiA6 (Ti plasmidwith octopine); ER: VRER gene.

FIG. 3: Effect of eutypine on the growth of grapevine calluses.GWT-eutypine: untransformed calluses cultured in the absence ofeutypine. GWT+eutypine: untransformned calluses cultured in the presenceof 500 μM of eutypine. GA, GB, GC, GD+eutypine: transformed callusescultured in the presence of 500 μM of eutypine.

DETAILED DESCRIPTION OF THE INVENTION

I--Demonstration of a Toxic Substance Synthesized by Eutypa lata,Eutine. Study of its Mode of Action and its Fate in Grapevine Cells.

The limited localization of the fungus in the trunk and the arms of thevarieties and the generalization of the symptoms throughout the aerialapparatus suggest that the fungus acts at a distance by the intermediaryof one or more toxic substances. This molecule was isolated andidentified, then its mode of action and its fate were studied ingrapevine cells.

Identification of a Toxic Substance Synthesized by E. lata ##STR1##

Among the 15 secondary metabolites synthesized in vitro by E. lata andcharacterized chemically, one molecule toxic for the grapevine wasdetected with the aid of various biotests. It is4-hydroxy-3-(3-methylbut-3-en-1-ynyl) benzaldehyde, called eutypine.

Several convergent facts have made it possible to show that eutypineparticipates in the expression of the symptoms of eutypa dieback in thegrapevine:

eutypine always proves to be absent from healthy grapevines and is foundin the sap, the leaves, the stems and the inflorescences of infectedplants. This result was obtained by using gas chromatography coupled intandem to a m a s s spectrophotometer (GC-MS-MS). However, this methodof identification has not made it possible to determine the eutypinepresent in the grapevine tissues;

eutypine reproduces certain symptoms of the disease when it is appliedto grapevine vitroplants;

eutypine induces ultrastructural alterations in the leaves ofvitroplants approximately similar to those observed on plants affectedby eutypa dieback: hypertrophy of the chloroplasts, dilation of thethylakoids, retraction of the plasmalemma, lysis of the cytoplasm,vesiculation of the endomembranes leading to the total deformation ofthe structure of the leaves of the cells;

the eutypine applied to simplified biological systems causes moreserious symptoms in the varieties sensitive to eutypa dieback than invarieties of grapevines tolerant to this disease. For example, theprotoplasts obtained from grapevine leaves of the Cabernet Sauvignonvariety exhibit a lower tolerance to eutypine than the protoplasts ofthe Merlot variety.

All of the data obtained lead to the conclusion that eutypine isimplicated in the appearance of the symptoms of the disease. At thelevel of the plant, it is synthesized by the mycelium in spring andtransported by the sap, it migrates towards the herbaceous organs. Thevariation of the quantity of eutypine synthesized might explain, forexample, the fact that a variety of grapevine harbouring the parasiticfungus does or does not exhibit symptoms.

The investigation of the mode of action of eutypine in the grapevinecells was made possible owing to the availability of eutypine in theunlabelled form and in the radioactive form, labelled with carbon 14.

It has been demonstrated that eutypine, a lipophilic weak acid,penetrates into the grapevine cells by passive diffusion. This toxicmolecule then accumulates in the cytoplasm by an acid trapping mechanismand, owing to its lipophilic character, inserts itself into the lipidsof the membranes.

Eutypine affects the functioning of the membrane systems of thegrapevine cells. It causes the acidification of the cytoplasm, thereduction of leucine transport, the activation at low dose of theconsumption of oxygen and its inhibition above 160 μM, and a diminutionof the energy load of the cells. These results strongly suggest thateutypine acts a mobile transporter of protons (protonophore).

The mode of action of eutypine was confirmed by investigating itseffects on the functioning of mitochondria. Eutypine acts as adecoupling agent of oxidative phosphorylation. The complete decouplingof the mitochondria is attained at concentrations of eutypine close to150 μM. The decoupling effect of eutypine is due to a protonophoreeffect linked to the dissociable alcohol function of the molecule sincea structural analogue which bears a methyl function in the place of thedissociable function has only a very weak effect on the functioning ofthe mitochondria.

All of these data demonstrate that eutypine acts as a mobile transporterof protons. The protonophore activity of eutypine may, on its own,account for the physiological adverse effects caused by the toxicmolecule.

Metabolism of Eugypine in Grapevine Cells

Grapevine cells, placed in the presence of eutypine (C¹⁴), produce aradioactive compound identified by mass spectrometry. It is ahydroxylated derivative of eutypine,4-hydroxy-3-(3-methylbut-3-en-1-ynyl) benzylic alcohol, calledeutypinol.

This compound does not show toxicity to the grapevine, even at highconcentrations, because it does not possess protonophore activity (TableI).

                  TABLE I                                                         ______________________________________                                        Effect of eutypinol on the viability of protoplasts obtained from              leaves of vitroplants of Vitis vinifera cv. Cabernet Sauvignon                      Treatment                                                                              Viability after 48 hours                                      ______________________________________                                        Control     88                                                                  Eutypine                                                                      100 μM 67                                                                  200 μM  0                                                                  Eutypinol                                                                     200 μM 86                                                                  500 μM 83                                                                ______________________________________                                    

The protoplasts (4.5×10⁵ /ml) are cultured in the presence of eutypineor eutypinol for 48 hours.

In order to demonstrate a relationship between the capacity tometabolize eutypine by the grapevine tissues and the behaviour of thedifferent varieties of grapevine towards eutypa dieback, thedetoxification activities of the protoplasts of leaves of the grapevinevarieties Ugni blanc (very sensitive), Cabernet Sauvignon (sensitive)and Merlot (tolerant) were compared (Table II).

                  TABLE II                                                        ______________________________________                                        Degradation of eutypine by protoplasts of different varieties of                Vitis vinifera                                                                                             Detoxification                                    Sensitivity to eutypa activity                                               Varieties dieback in vineyard (pmol/min/10.sup.5 cells)                     ______________________________________                                        Ugni blanc  very sensitive 7.9                                                  Cabernet Sauvignon sensitive 10.1                                             Merlot tolerant 13.6                                                        ______________________________________                                    

The protoplasts (10⁶ /ml) obtained from leaves of vitroplants areincubated for 1 hour at 30° C. in the presence of eutypine (100 μM; 0.4kBq). Then, the phenylacetylenic compounds are extracted, separated bythin layer chromatography and the metabolic rate of eutypine iscalculated after determination of the radioactivity associated witheutypine and eutypinol.

It is evident that the protoplasts of the varieties used in theexperiments can convert eutypine into eutypinol. The variety Merlot,considered to be tolerant to eutypa dieback in the grapevineyard,possesses a detoxification activity very much greater than that of Ugniblanc, a variety very sensitive to the disease. Furthermore, thedetoxification activity of the variety Cabernet Sauvignon, a varietywhich is also sensitive, is less than that of the variety Merlot.

These data clearly show the existence of a relationship between theability of these varieties to metabolize eutypine and their behaviour inthe vineyard with regard to eutypa dieback

The conversion of eutypine to eutypinol is catalyzed by a non-membraneenzyme, which specifically requires NADPH as co-factor. Its activity isreduced by reductase inhibitors such as disulfiram andparahydroxymercuribenzoic acid, confirming its membership of this groupof enzymes. It has been called "eutypine reductase". The reaction schemeis shown below. ##STR2## II. Investigation of a Gene Coding for aProtein Exhibiting a "Eutypine Reductase" Activity in a HeterologousSystems.

The objective was to isolate a gene coding for a protein exhibiting a"eutypine reductase" activity. It is recognized that the overexpresssionof a gene of a given species in this same species often leads to anoverall diminution of the expression of the endogenous gene and of thetransgene. On the other hand, the overexpression of a heterologous gene(belonging to a species different from that which is transformed) doesnot cause this phenomenon.

In order to avoid the phenomenon of co-suppression, a search was madeamong the different species of grapevine for the presence of an enzymecapable of detoxifying eutypine. Among the species studied, Vignaradiata (VR) showed the highest capacity to reduce eutypine. Theisolation of the protein was thus performed starting from the tissues ofthis species.

Purification of the ER Protein

The protein exhibiting a "eutypine reductase" activity was purified tohomogeneity according to the following protocol, then sequenced.

The seeds of Mung Bean (Vigna radiata L. R. Wilcz) were soaked overnightin aerated water. The seeds were then rinsed with water and sown onvermiculite. The plantlets were harvested after 4 days of culture at 23°C. and the hypocotyls (2 cm) were cut for the extraction of the enzyme.

Five hundred grams of hypocotyls of Mung Bean were ground in liquidnitrogen in a bead grinder of the Dangoumea type. One hundred grams ofpowder obtained were homogenized in 2 volumes of extraction buffercomposed of 0.1M of K-phosphate (pH 8.0), 10% of glycerol (wt/v), 1% ofpolyvinylpyrrolidone M 40,000, 30 mM Na ascorbate and 5 mM ofdithiothreitol (DTT). The homogenate was centrifuged for 20 min at48,000 g. The supernatant thus obtained was subjected to a doubleprecipitation with ammonium sulfate at 30 and 70% saturation. The pelletobtained from the precipitation with 70% ammonium sulfate was dissolvedin a small volume of 0.1 M K-phosphate buffer (pH 8.0) containing 10% ofglycerol and 1 mM of DTT. The enzymatic extract was desalted on a columnof Sephadex G25 (PD 10, Pharmacia), then purified in 6 successive steps:

hydrophobic interaction chromatography on a column of Phenyl-SepharoseCL4B (Pharmacia);

ionic interaction chromatography on a column of hydroxylapatite(Econo-Pac HTP, BioRad);

molecular exclusion chromatography on a column of Superose 12 HR of typeFPLC (Pharmacia);

ion exchange chromatography on a MonoQ 5/5 column of FPLC type(Pharmacia);

chromatography by high performance electrophoresis (HPEC) on native gel

chromatography by denaturing electrophoresis (SDS-PAGE).

Alternatively, the ER protein could be purified from Mung Bean seeds byapplying the following procedure:

Five hundred grams of hypocotyls of Mung Bean were ground in a propellergrinder in the presence of two volumes of extraction buffer composed of0.1 M of borate buffer (pH 8.0), 10% of glycerol (wt/v), 1% ofpolyvinylpyrrolidine 40,000 and 4 mM of dithiothreitol (DTT). Thehomogenate was centrifuged for 20 min at 48,000 g. The supernatant thusobtained was subjected to a double precipitation with ammonium sulfateat 30 and 70% of saturation. The pellet obtained from the precipitationwith 70% ammonium sulfate was dissolved in a small volume of 25 mMK-phosphate buffer (pH 8.0) containing 10% of glycerol and 1 M ofammonium sulfate. The enzymatic extract was purified in 5 successivesteps:

hydrophobic interaction chromatography on a column of Phenyl-SepharoseCL4B (Pharmacia);

ionic interaction chromatography on a column of hydroxylapatite(Econo-Pac HTP, BioRad);

molecular exclusion chromatography on a column of Superose 12 HR of typeFPLC (Pharmada);

ion exchange chromatography on a MonoQ 5/5 column of FPLC type(Pharmacia);

chromatography by denaturing electrophoresis (SDS-PAGE).

The protein thus purified to homogeneity by SDS-PAGE was hydrolysedovernight at 30° C. by a lysine endoprotease and the peptides releasedwere separated by HPLC on a reverse phase DEAE-C18 column. Five of theseparated peptides were sequenced with the aid of an Applied Biosystems470 microsequencer.

Isolation and Nucleotide Sequence of the VRER Gene

With the aid of degenerate primers derived from these peptide sequences,a DNA fragment was amplified by the PCR procedure ("polymerase chainreaction"). A reverse PCR then made it possible to isolate the 3' and 5'parts of this clone. Finally, the full length clone was then isolated bychoosing the primers suitable for the 3' and 5' ends and was sequencedseveral times. The complementary DNA possesses an open reading frame of975 nucleotides. The amino add sequence deduced indicates that the clonecodes for a polypeptide composed of 325 amino acids. The size of theprotein is estimated at 36 kD and has an expected pI of 6.34. Thesequence of the VRER gene exhibits homologies (between 20 and 26% ofidentity at the amino acid level) with the proteins of the family of thedihydroflavonol reductases, and with the HC toxin of maize.

In order to verify that the protein encoded in this gene exhibits a"eutypine reductase" activity and is capable of reducing eutypine, thisprotein was overexpressed in a bacterial system, Escherichia coli whichis incapable of reducing eutypine in the natural state. For that, thecomplementary DNA of the VRER gene was cloned in an expression vector,then transferred into the bacteria. The lysate of these bacteria whichoverexpress the protein corresponding to the VRER gene uses theco-factor NADPH to reduce eutypine. The production of eutypinol wasdemonstrated by thin layer chromatography of a bacterial lysateincubated in the presence of radioactive eutypine. All of these datashow that the protein corresponding to the VRER gene exhibits a"eutypine reductase" activity in vitro.

Biochemical Characteristics of the Protein Encoded in the VRER Gene.

The protein encoded in the VRER gene was characterized biochemically. Ithas a Km of 16.4 M for eutypine, which represents an excellent affinityof this enzyme for the substrate eutypine. It was also investigatedwhether this protein was capable of reducing other substrates. Thefamily of the benzaldehydes (benzaldehyde and some of its derivativessuch as methylbenzaldehyde, methoxybenzaldehyde, nitrobenzaldehyde,fluorobenzaldehyde possessing a group in the meta and para position),linear aldehydes such as decylaldehyde or hexanal, or other cycliccompounds possessing an aldehyde function like 4pyridine carbaldehyde or2-fluoraldehyde are also reduced by the ER protein in the presence ofNADPH.

III--Study of the Expression of the VRER Gene in Grapevine Cells

The objective of this study was to verify whether the expression of theVRER gene in grapevine cells is capable of increasing their capacity tometabolize eutypine to eutypinol. For that, the VRER gene was introducedinto a binary transformation vector, then integrated into the callusesof the grapevine cv Gamay. The transformed calluses were then confrontedwith eutypine in order to analyze their behaviour and to determine thelevel of resistance of the plant to eutypine.

Integration of the VRER Gene in a Binary Transformation Vector

The VRER gene was integrated into the binary plasmid pGA (An G et al.,Binary vectors. In Plant Molecular Biology Manual A3. Kluwer Acad. Pub.,Dordrecht, The Nederlands, 1-19) between the right (RB) and the left(LB) borders. This binary vector contains the genes for resistance tokanamycin and tetracycline. The gene of interest was cloned into theT-DNA, behind the 35S promoter of the CaMV which enables it to beexpressed in plant cells.

The schematic representation of the binary vector pGA-ER thus obtainedis given in FIG. 2.

The plasmid pGA-ER was introduced into Agrobacterium tumefaciens strainC58. The presence of this plasmid in the bacteria transformed onselective medium (kanamycin 10 mg/l and tetracycline 5 mg/l) wasconfirmed by PCR analysis. The C58 strain of Agrobacterium tumefacienscontaining the plasmid pGA-ER is then designated AbC58ER. It will beused subsequently to carry out transformation experiments on calluses ofVitis vinifera cv Gamay.

Transformation of the Grapevine Calluses via Agrobacterium tumefaciens

The transformation experiments were carried out on calluses of Vitisvinifera cv Gamay, obtained from epidermal cells of the fruit.

The genetic transformation of the calluses of Vitis vinifera cv Gamaywas carried out via the Agrobacterium tumefaciens strain AbC58-ER,containing the binary vector pAG-ER.

The calluses, cultured for 8 days in a 55 mm Petri dish (10 calluses perdish), were immersed in the bacterial suspension for 20 minutes. Theinoculum was then removed by aspiration and the calluses were dried for5 minutes in the hood. They were then transferred to a new medium, onthe top of which a paper disk was placed, and cultured in the dark at atemperature of 28° C. At the end of a o-culture for 48 hours, thecalluses were rinsed carefully with culture medium containing 400 mg/lof carbenicilin (antibiotic intended to eliminate the bacteria), driedon blotting paper and transferred to a new medium with 400 mg/l ofcarbenicillin. The selection pressure was applied 7 days afterinoculation: the calluses were transferred to a selective "Gamay" mediumcontaining a selective antibiotic (kanamycin at 75 mg/l), in addition tocarbenicillin.

The calluses were maintained on the "Gamay" medium, subcultured every 20days on fresh selective medium and placed in a culture chamber at 24°C., with an illumination of 100 μmol.m⁻². sec⁻¹ and a photoperiod of 16hours.

Three months later, after 4 successive subcultures on selective newmedium, calluses tolerant to kanamycin were obtained. They were thenmaintained on selective culture media containing 75 mg/l of kanamycin.

The verification of the integration of the VRER gene in the genome ofthe grapevine calluses expressing marked resistance to kanamycin wasperformed by the PCR (polymerase chain reaction) method and by molecularhybridization (Southern blot). Finally, the level of expression of thetransgene was determined by Western blot, with the aid of antibodiesdirected against the ER protein.

Analysis of the Behaviour of Transformed Grapevine Calluses TowardsEutypine

The genetically transformed grapevine calluses and expressing the VRERtransgene have then been confronted with eutypine to determine the levelof resistance acquired by these tissues to eutypine.

Two biotests were used. The first (biotest I) is based on theconfrontation of grapevine cells cultured in suspension with eutypineapplied at different concentrations. The second makes use of grapevinecalluses cultured on culture media containing eutypine.

In the biotest I, the percentage of dead cells was estimated by means ofthe erythrosine reagent. The concentration of eutypine inducing apercentage of dead cells of 50% is determined in the wildtype strain andin the transformed varieties. The results presented in Table III showthat the cells of the transformed grapevine varieties exhibit an LD 50 2to 2.5 times higher than that recorded for the untransformed wildtypevariety.

Table III.

Eutypine concentration causing the mortality of 50% (LD50) of thetransformed (GA, GB, GC, GD) or untransformed (GWT) grapevine cells,grown in suspension in the presence of this toxic molecule.

    ______________________________________                                                   Cellular grapevine varieties                                                    GWT     GA      GB    GC    GD                                   ______________________________________                                        LD50 (μM of eutypine)                                                                   100     255     250   230   225                                  ______________________________________                                         GWT: wildtype variety, untransformed.                                         GA, GB, GC, GD: transformed grapevine strains                            

The biotest II reveals that the transformed grapevine calluses haveacquired a level of resistance to eutypine very much greater than thecapacity of the untransformed calluses. The results presented below showthat the calluses of the wildtype variety do not develop in the presenceof 500 μM of eutypine and necrose very rapidly. On the other hand, theGA variety, grown in the presence of eutypine supplied at aconcentration which is usually lethal for these tissues, does notexhibit necroses and shows a growth practically similar to that of thecontrol tissues which develop in the absence of eutypine (FIG. 3).

All of these results indicate that the introduction and expression ofthe VRER gene in the grapevine cells confer a greater resistance to thistoxic molecule. It is due to the overproduction in the transformedgrapevine cells of the ER protein, capable of metabolizing eutypine toeutypinol, a molecule non-toxic for the grapevine. These data show theefficacy of this gene for the detoxification of eutypine.

The transformation experiments were also performed on calluses of Vitisvinifera cv Ugni blanc by making use of the same experimental protocolas that used for the transformation of the calluses of Vitis viniferacv. Gamay.

The analysis of the behaviour of the calluses of Vitis vinifera cv Ugniblanc towards eutypine was performed by using biotest II. The resultspresented below show that the grapevine calluses have acquired a levelof resistance to eutypine very much higher than that of theuntransformed calluses. In the presence of 500 μM, the calluses of thewildtype variety necrose rapidly and do not develop. At thisconcentration, the growth of the calluses of the transformed UB varietyis practically unaffected (Table IV). These results demonstrate theefficacy of this gene for detoxifying eutypine in the grapevine tissues.

Table IV.

Development of the transformed (UBT) and untransformed (UBWT) callusesof Vitis vinifera cv Ugni blanc cultured in the presence of 500 μM ofeutypine.

    ______________________________________                                                     Mean mass of a callus                                              Cell varieties (mg)                                                         ______________________________________                                        UBWT          25                                                                UBT 150                                                                     ______________________________________                                    

IV--Preparation of Antibodies Directed Against the Protein Encoded inthe VRER Gene.

In order to obtain antibodies directed against this protein, thefollowing procedure was used.

1.1--Cloning of the VRER Gene

The cDNA fragment bearing the active part of the VRER gene was clonedinto the vector pT7.7 (Tabor S et al., 1985, Proc. Natl. Acad. Sci. USA,82, 1047-1078) at the EcoRI sites. It was then introduced in E.coli.After having confirmed by Southern blot that the plasmid has beencorrectly integrated, it was verified that the bacterial strainsselected produce the ER protein capable of metabolizing eutypine intoeutypinol.

1.2--Production and Partial Purification of the Recombinant Protein

The bacteria carrying the VRER transgene were placed at 42° C. for 20minutes, then the production of the recombinant protein was allowed toproceed at 37° C. for 1 hour. After centrifugation, the bacteria wereresuspended in a 0.1 M Tris HCl buffer (pH 8.0), 1 mM EDTA, 0.1 M NaClsupplemented with lysozyme (1 mg/ml). After incubation at 37° C. for 15minutes, the solution was treated with 0.2 mg/ml of DNAse in thepresence of PMSF (0.2 mM) and MgCl₂ (4 mM) for 15 minutes. Aftercentrifugation at 12,000 g for 10 minutes at 4° C., the protein pelletcontaining the recombinant protein was washed with a 3M urea solution.After centrifugation, the protein extract was denatured in the 0.1 MTris HCl buffer (pH 7.5) containing urea (6M). The denatured proteinswere then concentrated 5 times (Centricon 10).

1.3--Immunization of Rabbits by the Protein Extract Containing theRecombinant Protein.

An aliquot (50 μg) of the protein extract containing the recombinantprotein was fractionated by preparative electrophoresis (12%polyacrylamide gel in the presence of SDS). The proteins were thentransferred to a nitrocellulose membrane and the recombinant protein waslocated by staining with Coomassie blue. A band of nitrocellulosebearing the recombinant protein was cut out and used as implant for theimmunization of two rabbits (Eurogentec company). Four implantationswere made during the first two months of immunization and the finallybleeding was done 1 month after the last immunization.

1.4--Partial Purification of the Antibodies Directed Against the ERProtein.

The anti-ER protein antibodies were partially purified by exhaustion ofthe rabbit serum against the total proteins of E. coli. The proteinsextracted from wildtype E. coli are deposited on a nitrocellulosemembrane, then this latter is placed in the serum obtained from therabbits. This operation is repeated several times until the serum isexhausted.

After verification, the serum thus obtained is used in Western blotanalyses.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 4                                           - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 975 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..975                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - ATG AGC ACC GCC GCT GGA AAA CTA GTC TGT GT - #C ACC GGC GCT TCC        GGT       48                                                                    Met Ser Thr Ala Ala Gly Lys Leu Val Cys Va - #l Thr Gly Ala Ser Gly            1               5 - #                 10 - #                 15              - - TAC ATC GCT TCC TGG CTC GTC AAG TTT CTT CT - #C GAA CGC GGC TAC ACC           96                                                                       Tyr Ile Ala Ser Trp Leu Val Lys Phe Leu Le - #u Glu Arg Gly Tyr Thr                        20     - #             25     - #             30                  - - GTG AAG GCC ACC GTT CGC GAC ACG AAT GAT CC - #C AAA AAG GTA GAT CAC          144                                                                       Val Lys Ala Thr Val Arg Asp Thr Asn Asp Pr - #o Lys Lys Val Asp His                    35         - #         40         - #         45                      - - TTG CTC AGC CTT GAT GGT GCG AAG GAG AGA TT - #G CAT CTC GTG AAG GCG          192                                                                       Leu Leu Ser Leu Asp Gly Ala Lys Glu Arg Le - #u His Leu Val Lys Ala                50             - #     55             - #     60                          - - AAT CTT CTA GAA GAA GGT TCC TTT GAC TCT GC - #C GTT GAA GGT GTT CAC          240                                                                       Asn Leu Leu Glu Glu Gly Ser Phe Asp Ser Al - #a Val Glu Gly Val His            65                 - # 70                 - # 75                 - # 80       - - GCT GTG TTT CAC ACT GCT TCT CCC TTT TTC AA - #C GAT GCC AAA GAT CCG          288                                                                       Ala Val Phe His Thr Ala Ser Pro Phe Phe As - #n Asp Ala Lys Asp Pro                            85 - #                 90 - #                 95              - - CAG ACT GAG TTG TTG GAC CCG GCA GTG AAG GG - #G ACT CTG AAT GTT CTG          336                                                                       Gln Thr Glu Leu Leu Asp Pro Ala Val Lys Gl - #y Thr Leu Asn Val Leu                       100      - #           105      - #           110                  - - AAA TCG TGT GTG AAC TCG CCC ACG CTG AAA CG - #C GTC GTT TTA ACT TCT          384                                                                       Lys Ser Cys Val Asn Ser Pro Thr Leu Lys Ar - #g Val Val Leu Thr Ser                   115          - #       120          - #       125                      - - TCT ATT GCT GCA GTT GCG TTC AGC GAC AGG CC - #T AAA AAC CCT GAT GTT          432                                                                       Ser Ile Ala Ala Val Ala Phe Ser Asp Arg Pr - #o Lys Asn Pro Asp Val               130              - #   135              - #   140                          - - GTG GTT GAC GAG ACT TGG TAT TCT GAC CCG GA - #A TAC TGT AAG AGA ACA          480                                                                       Val Val Asp Glu Thr Trp Tyr Ser Asp Pro Gl - #u Tyr Cys Lys Arg Thr           145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - GGG TTA TGG TAT AAC CTT TCA AAG ACT CTG GC - #C GAA GAT GCT GCC        TGG      528                                                                    Gly Leu Trp Tyr Asn Leu Ser Lys Thr Leu Al - #a Glu Asp Ala Ala Trp                          165  - #               170  - #               175              - - AAA TTT GCG AAA GAA AAC AAC ATT GAC CTG GT - #T ACA ATG AAC CCA GCA          576                                                                       Lys Phe Ala Lys Glu Asn Asn Ile Asp Leu Va - #l Thr Met Asn Pro Ala                       180      - #           185      - #           190                  - - TTG GTT GTT GGA CCT CTC TTG CAA CCA GTG CT - #T AAT ACT AGC GCT GCT          624                                                                       Leu Val Val Gly Pro Leu Leu Gln Pro Val Le - #u Asn Thr Ser Ala Ala                   195          - #       200          - #       205                      - - ATA GTT TTA GGT TTA GTT AAT GGG GCG AAA AC - #A TTT AAA AAT GCT TCT          672                                                                       Ile Val Leu Gly Leu Val Asn Gly Ala Lys Th - #r Phe Lys Asn Ala Ser               210              - #   215              - #   220                          - - TTG GGA TGG GTC GAC GTG AAA GAT GTT GCA TT - #G GCC CAT ATT CTA GCA          720                                                                       Leu Gly Trp Val Asp Val Lys Asp Val Ala Le - #u Ala His Ile Leu Ala           225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - TAT GAG AAT GCT TCG GCT AAT GGA AGA TAT TT - #A CTA GTT GAG AGA        GTA      768                                                                    Tyr Glu Asn Ala Ser Ala Asn Gly Arg Tyr Le - #u Leu Val Glu Arg Val                          245  - #               250  - #               255              - - GCA CAC TTT GGA GAT GCT GCC AAG ATT TTA CG - #A GAT TTA TAC CCA ACA          816                                                                       Ala His Phe Gly Asp Ala Ala Lys Ile Leu Ar - #g Asp Leu Tyr Pro Thr                       260      - #           265      - #           270                  - - TTG CAA ATT CCA GAC AAG TGT GAA GAC GAT AA - #G CCA TTA GAA CCA ATA          864                                                                       Leu Gln Ile Pro Asp Lys Cys Glu Asp Asp Ly - #s Pro Leu Glu Pro Ile                   275          - #       280          - #       285                      - - TTT CAG GTT TCG AAG GAA AAA GCA AAG AGC TT - #G GGG ATT GAC TAT ATT          912                                                                       Phe Gln Val Ser Lys Glu Lys Ala Lys Ser Le - #u Gly Ile Asp Tyr Ile               290              - #   295              - #   300                          - - CCT TTG GAA GTG AGC CTC AAG GAC ACT GTG GA - #G AGC TTG AAG GAA AAG          960                                                                       Pro Leu Glu Val Ser Leu Lys Asp Thr Val Gl - #u Ser Leu Lys Glu Lys           305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - AAG TTT CTG AAA GTT           - #                  - #                      - #   975                                                                 Lys Phe Leu Lys Val                                                                           325                                                            - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 325 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - Met Ser Thr Ala Ala Gly Lys Leu Val Cys Va - #l Thr Gly Ala Ser Gly        1               5 - #                 10 - #                 15              - - Tyr Ile Ala Ser Trp Leu Val Lys Phe Leu Le - #u Glu Arg Gly Tyr Thr                   20     - #             25     - #             30                  - - Val Lys Ala Thr Val Arg Asp Thr Asn Asp Pr - #o Lys Lys Val Asp His               35         - #         40         - #         45                      - - Leu Leu Ser Leu Asp Gly Ala Lys Glu Arg Le - #u His Leu Val Lys Ala           50             - #     55             - #     60                          - - Asn Leu Leu Glu Glu Gly Ser Phe Asp Ser Al - #a Val Glu Gly Val His       65                 - # 70                 - # 75                 - # 80       - - Ala Val Phe His Thr Ala Ser Pro Phe Phe As - #n Asp Ala Lys Asp Pro                       85 - #                 90 - #                 95              - - Gln Thr Glu Leu Leu Asp Pro Ala Val Lys Gl - #y Thr Leu Asn Val Leu                  100      - #           105      - #           110                  - - Lys Ser Cys Val Asn Ser Pro Thr Leu Lys Ar - #g Val Val Leu Thr Ser              115          - #       120          - #       125                      - - Ser Ile Ala Ala Val Ala Phe Ser Asp Arg Pr - #o Lys Asn Pro Asp Val          130              - #   135              - #   140                          - - Val Val Asp Glu Thr Trp Tyr Ser Asp Pro Gl - #u Tyr Cys Lys Arg Thr      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Gly Leu Trp Tyr Asn Leu Ser Lys Thr Leu Al - #a Glu Asp Ala Ala        Trp                                                                                             165  - #               170  - #               175             - - Lys Phe Ala Lys Glu Asn Asn Ile Asp Leu Va - #l Thr Met Asn Pro Ala                  180      - #           185      - #           190                  - - Leu Val Val Gly Pro Leu Leu Gln Pro Val Le - #u Asn Thr Ser Ala Ala              195          - #       200          - #       205                      - - Ile Val Leu Gly Leu Val Asn Gly Ala Lys Th - #r Phe Lys Asn Ala Ser          210              - #   215              - #   220                          - - Leu Gly Trp Val Asp Val Lys Asp Val Ala Le - #u Ala His Ile Leu Ala      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Tyr Glu Asn Ala Ser Ala Asn Gly Arg Tyr Le - #u Leu Val Glu Arg        Val                                                                                             245  - #               250  - #               255             - - Ala His Phe Gly Asp Ala Ala Lys Ile Leu Ar - #g Asp Leu Tyr Pro Thr                  260      - #           265      - #           270                  - - Leu Gln Ile Pro Asp Lys Cys Glu Asp Asp Ly - #s Pro Leu Glu Pro Ile              275          - #       280          - #       285                      - - Phe Gln Val Ser Lys Glu Lys Ala Lys Ser Le - #u Gly Ile Asp Tyr Ile          290              - #   295              - #   300                          - - Pro Leu Glu Val Ser Leu Lys Asp Thr Val Gl - #u Ser Leu Lys Glu Lys      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Lys Phe Leu Lys Val                                                                      325                                                            - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1254 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 22..996                                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - GCAGAATCAA CATAATCCAC A ATG AGC ACC GCC GCT GGA - #AAA CTA GTC TGT           51                                                                                         - #      Met Ser Thr Ala Ala Gly Lys - #Leu Val Cys                           - #        1          - #     5             - #     10       - - GTC ACC GGC GCT TCC GGT TAC ATC GCT TCC TG - #G CTC GTC AAG TTT CTT           99                                                                       Val Thr Gly Ala Ser Gly Tyr Ile Ala Ser Tr - #p Leu Val Lys Phe Leu                            15 - #                 20 - #                 25              - - CTC GAA CGC GGC TAC ACC GTG AAG GCC ACC GT - #T CGC GAC ACG AAT GAT          147                                                                       Leu Glu Arg Gly Tyr Thr Val Lys Ala Thr Va - #l Arg Asp Thr Asn Asp                        30     - #             35     - #             40                  - - CCC AAA AAG GTA GAT CAC TTG CTC AGC CTT GA - #T GGT GCG AAG GAG AGA          195                                                                       Pro Lys Lys Val Asp His Leu Leu Ser Leu As - #p Gly Ala Lys Glu Arg                    45         - #         50         - #         55                      - - TTG CAT CTC GTG AAG GCG AAT CTT CTA GAA GA - #A GGT TCC TTT GAC TCT          243                                                                       Leu His Leu Val Lys Ala Asn Leu Leu Glu Gl - #u Gly Ser Phe Asp Ser                60             - #     65             - #     70                          - - GCC GTT GAA GGT GTT CAC GCT GTG TTT CAC AC - #T GCT TCT CCC TTT TTC          291                                                                       Ala Val Glu Gly Val His Ala Val Phe His Th - #r Ala Ser Pro Phe Phe            75                 - # 80                 - # 85                 - # 90       - - AAC GAT GCC AAA GAT CCG CAG ACT GAG TTG TT - #G GAC CCG GCA GTG AAG          339                                                                       Asn Asp Ala Lys Asp Pro Gln Thr Glu Leu Le - #u Asp Pro Ala Val Lys                            95 - #                100 - #                105              - - GGG ACT CTG AAT GTT CTG AAA TCG TGT GTG AA - #C TCG CCC ACG CTG AAA          387                                                                       Gly Thr Leu Asn Val Leu Lys Ser Cys Val As - #n Ser Pro Thr Leu Lys                       110      - #           115      - #           120                  - - CGC GTC GTT TTA ACT TCT TCT ATT GCT GCA GT - #T GCG TTC AGC GAC AGG          435                                                                       Arg Val Val Leu Thr Ser Ser Ile Ala Ala Va - #l Ala Phe Ser Asp Arg                   125          - #       130          - #       135                      - - CCT AAA AAC CCT GAT GTT GTG GTT GAC GAG AC - #T TGG TAT TCT GAC CCG          483                                                                       Pro Lys Asn Pro Asp Val Val Val Asp Glu Th - #r Trp Tyr Ser Asp Pro               140              - #   145              - #   150                          - - GAA TAC TGT AAG AGA ACA GGG TTA TGG TAT AA - #C CTT TCA AAG ACT CTG          531                                                                       Glu Tyr Cys Lys Arg Thr Gly Leu Trp Tyr As - #n Leu Ser Lys Thr Leu           155                 1 - #60                 1 - #65                 1 -      #70                                                                              - - GCC GAA GAT GCT GCC TGG AAA TTT GCG AAA GA - #A AAC AAC ATT GAC        CTG      579                                                                    Ala Glu Asp Ala Ala Trp Lys Phe Ala Lys Gl - #u Asn Asn Ile Asp Leu                          175  - #               180  - #               185              - - GTT ACA ATG AAC CCA GCA TTG GTT GTT GGA CC - #T CTC TTG CAA CCA GTG          627                                                                       Val Thr Met Asn Pro Ala Leu Val Val Gly Pr - #o Leu Leu Gln Pro Val                       190      - #           195      - #           200                  - - CTT AAT ACT AGC GCT GCT ATA GTT TTA GGT TT - #A GTT AAT GGG GCG AAA          675                                                                       Leu Asn Thr Ser Ala Ala Ile Val Leu Gly Le - #u Val Asn Gly Ala Lys                   205          - #       210          - #       215                      - - ACA TTT AAA AAT GCT TCT TTG GGA TGG GTC GA - #C GTG AAA GAT GTT GCA          723                                                                       Thr Phe Lys Asn Ala Ser Leu Gly Trp Val As - #p Val Lys Asp Val Ala               220              - #   225              - #   230                          - - TTG GCC CAT ATT CTA GCA TAT GAG AAT GCT TC - #G GCT AAT GGA AGA TAT          771                                                                       Leu Ala His Ile Leu Ala Tyr Glu Asn Ala Se - #r Ala Asn Gly Arg Tyr           235                 2 - #40                 2 - #45                 2 -      #50                                                                              - - TTA CTA GTT GAG AGA GTA GCA CAC TTT GGA GA - #T GCT GCC AAG ATT        TTA      819                                                                    Leu Leu Val Glu Arg Val Ala His Phe Gly As - #p Ala Ala Lys Ile Leu                          255  - #               260  - #               265              - - CGA GAT TTA TAC CCA ACA TTG CAA ATT CCA GA - #C AAG TGT GAA GAC GAT          867                                                                       Arg Asp Leu Tyr Pro Thr Leu Gln Ile Pro As - #p Lys Cys Glu Asp Asp                       270      - #           275      - #           280                  - - AAG CCA TTA GAA CCA ATA TTT CAG GTT TCG AA - #G GAA AAA GCA AAG AGC          915                                                                       Lys Pro Leu Glu Pro Ile Phe Gln Val Ser Ly - #s Glu Lys Ala Lys Ser                   285          - #       290          - #       295                      - - TTG GGG ATT GAC TAT ATT CCT TTG GAA GTG AG - #C CTC AAG GAC ACT GTG          963                                                                       Leu Gly Ile Asp Tyr Ile Pro Leu Glu Val Se - #r Leu Lys Asp Thr Val               300              - #   305              - #   310                          - - GAG AGC TTG AAG GAA AAG AAG TTT CTG AAA GT - #T TAATGTTACC CTTCAAGAA    A   1016                                                                       Glu Ser Leu Lys Glu Lys Lys Phe Leu Lys Va - #l                               315                 3 - #20                 3 - #25                            - - CGTGGAAACC TTCTTATGCC TATGCTTGAG GTAAACTTAG TGTTAAATAA AT -             #GGAAAGTG   1076                                                                 - - ATGTAAGTTC TGTAATAAGT GATTCTTGGA CTGGTTTTTT CTGGTGACAA GT -            #TTCAAAAA   1136                                                                 - - TGAGTGACCA TGGTAGAGTC TGCGTTCCAG ATTGTATCCT TGTGATATTG TA -            #TTAGTAGA   1196                                                                 - - AGATGTTTGT TGCAGCTAAT GCACCCATTA TTATGTTTTT ATCAAAAAAA AA -            #AAAAAA     1254                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 325 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - Met Ser Thr Ala Ala Gly Lys Leu Val Cys Va - #l Thr Gly Ala Ser        Gly                                                                               1               5 - #                 10 - #                 15             - - Tyr Ile Ala Ser Trp Leu Val Lys Phe Leu Le - #u Glu Arg Gly Tyr Thr                   20     - #             25     - #             30                  - - Val Lys Ala Thr Val Arg Asp Thr Asn Asp Pr - #o Lys Lys Val Asp His               35         - #         40         - #         45                      - - Leu Leu Ser Leu Asp Gly Ala Lys Glu Arg Le - #u His Leu Val Lys Ala           50             - #     55             - #     60                          - - Asn Leu Leu Glu Glu Gly Ser Phe Asp Ser Al - #a Val Glu Gly Val His       65                 - # 70                 - # 75                 - # 80       - - Ala Val Phe His Thr Ala Ser Pro Phe Phe As - #n Asp Ala Lys Asp Pro                       85 - #                 90 - #                 95              - - Gln Thr Glu Leu Leu Asp Pro Ala Val Lys Gl - #y Thr Leu Asn Val Leu                  100      - #           105      - #           110                  - - Lys Ser Cys Val Asn Ser Pro Thr Leu Lys Ar - #g Val Val Leu Thr Ser              115          - #       120          - #       125                      - - Ser Ile Ala Ala Val Ala Phe Ser Asp Arg Pr - #o Lys Asn Pro Asp Val          130              - #   135              - #   140                          - - Val Val Asp Glu Thr Trp Tyr Ser Asp Pro Gl - #u Tyr Cys Lys Arg Thr      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Gly Leu Trp Tyr Asn Leu Ser Lys Thr Leu Al - #a Glu Asp Ala Ala        Trp                                                                                             165  - #               170  - #               175             - - Lys Phe Ala Lys Glu Asn Asn Ile Asp Leu Va - #l Thr Met Asn Pro Ala                  180      - #           185      - #           190                  - - Leu Val Val Gly Pro Leu Leu Gln Pro Val Le - #u Asn Thr Ser Ala Ala              195          - #       200          - #       205                      - - Ile Val Leu Gly Leu Val Asn Gly Ala Lys Th - #r Phe Lys Asn Ala Ser          210              - #   215              - #   220                          - - Leu Gly Trp Val Asp Val Lys Asp Val Ala Le - #u Ala His Ile Leu Ala      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Tyr Glu Asn Ala Ser Ala Asn Gly Arg Tyr Le - #u Leu Val Glu Arg        Val                                                                                             245  - #               250  - #               255             - - Ala His Phe Gly Asp Ala Ala Lys Ile Leu Ar - #g Asp Leu Tyr Pro Thr                  260      - #           265      - #           270                  - - Leu Gln Ile Pro Asp Lys Cys Glu Asp Asp Ly - #s Pro Leu Glu Pro Ile              275          - #       280          - #       285                      - - Phe Gln Val Ser Lys Glu Lys Ala Lys Ser Le - #u Gly Ile Asp Tyr Ile          290              - #   295              - #   300                          - - Pro Leu Glu Val Ser Leu Lys Asp Thr Val Gl - #u Ser Leu Lys Glu Lys      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Lys Phe Leu Lys Val                                                                      325                                                         __________________________________________________________________________

What is claimed is:
 1. An isolated nucleotide sequence which codes for apolypeptide with eutypine reductase activity, said polypeptidecomprising the following amino acid sequence (SEQ ID NO.2):

    ______________________________________                                        Met Ser Thr Ala Ala Gly Lys Leu Val                                             Cys Val Thr Gly Ala Ser Gly Tyr Ile                                         Ala Ser Trp Leu Val Lys Phe Leu Leu                                             Glu Arg Gly Tyr Thr Val Lys Ala Thr                                            -  Val Arg Asp Thr Asn Asp Pro Lys Lys                                               Val Asp His Leu Leu Ser Leu Asp                                        -        Gly Ala Lys Glu Arg Leu His Leu Val                                             Lys Ala Asn Leu Leu Glu Glu Gly Ser                                -            Phe Asp Ser Ala Val Glu Gly Val His                                             Ala Val Phe His Thr Ala Ser Pro Phe                            -                Phe Asn Asp Ala Lys Asp Pro Gln Thr                                             Glu Leu Leu Asp Pro Ala Val Lys                            -                    Gly Thr Leu Asn Val Leu Lys Ser Cys                                             Val Asn Ser Pro Thr Leu Lys Arg Val                    -                                                                          Val Leu Thr Ser Ser Ile Ala Ala Val                                             Ala Phe Ser Asp Arg Pro Lys Asn Pro                                            -  Asp Val Val Val Asp Glu Thr Trp Tyr                                               Ser Asp Pro Glu Tyr Cys Lys Arg Thr                                    -        Gly Leu Trp Tyr Asn Leu Ser Lys Thr                                             Leu Ala Glu Asp Ala Ala Trp Lys Phe                                -            Ala Lys Glu Asn Asn Ile Asp Leu Val                                             Thr Met Asn Pro Ala Leu Val Val Gly                            -                Pro Leu Leu Gln Pro Val Leu Asn Thr                                             Ser Ala Ala Ile Val Leu Gly Leu Val                        -                    Asn Gly Ala Lys Thr Phe Lys Asn Ala                                             Ser Leu Gly Trp Val Asp Val Lys Asp                    -                                                                          Val Ala Leu Ala His Ile Leu Ala Tyr                                             Glu Asn Ala Ser Ala Asn Gly Arg Tyr                                            -  Leu Leu Val Glu Arg Val Ala His Phe                                               Gly Asp Ala Ala Lys Ile Leu Arg Asp                                    -        Leu Tyr Pro Thr Leu Gln Ile Pro Asp                                             Lys Cys Glu Asp Asp Lys Pro Leu Glu                                -            Pro Ile Phe Gln Val Ser Lys Glu Lys                                             Ala Lys Ser Leu Gly Ile Asp Tyr Ile Pro                        -                Leu Glu Val Ser Leu Lys Asp Thr Val                                             Glu Ser Leu Lys Glu Lys Lys Phe Leu                        -                    Lys Val.                                              ______________________________________                                    


2. The isolated nucleotide sequence according to claim 1 comprising thefollowing nucleotide sequence (SEQ ID NO.1):

    ______________________________________                                        ATGAGCACCGCCGCTGGAAAACTAGTCTGTGTCACCGGCGCTTCCGG                               TTACATCGCTTCCTGGCTCGTCAAGTTTCTTCTCGAACGCGGCTACAC                                 - CGTGAAGGCCACCGTTCGCGACACGAATGATCCCAAAAAGGTAGATC                             - ACTTGCTCAGCCTTGATGGTGCGAAGGAGAGATTGCATCTCGTGAAG                             - GCGAATCTTCTAGAAGAAGGTTCCTTTGACTCTGCCGTTGAAGGTGTT                            - CACGCTGTGTTTCACACTGCTTCTCCCTTTTTCAACGATGCCAAAGATC                           - CGCAGACTGAGTTGTTGGACCCGGCAGTGAAGGGGACTCTGAATGTT                             - CTGAAATCGTGTGAACTCGCCCACGCTGAAACGCGTCGTTTTAACT                              - TCTTCTATTGCTGCAGTTGCGTTCAGCGACAGGCCTAAAAACCCTGAT                            - GTTGTGGTTGACGAGACTTGGTATTCTGACCCGGAATACTGTAAGAGA                            - ACAGGGTTATGGTATAACCTTTCAAAGACTCTGGCCGAAGATGCTGCC                            - TGGAAATTTGCGAAAGAAAACAACATTGACCTGGTTACAATGAACCCA                            - GCATTGGTTGTTGGACCTCTCTTGCAACCAGTGCTTAATACTAGCGCT                            - GCTATAGTTTTAGGTTTAGTTAATGGGGCGAAAACATTTAAAAATGCTT                           - CTTTGGGATGGGTCGACGTGAAAGATGTTGCATTGGCCCATATTCTAG                            - CATATGAGAATGCTTCGGCTAATGGAAGATATTTACTAGTTGAGAGAG                            - TAGCACACTTTGGAGATGCTGCCAAGATTTTACGAGATTTATACCCAAC                           - ATTGCAAATTCCAGACAAGTGTGAAGACGATAAGCCATTAGAACCAAT                            - ATTTCAGGTTTCGAAGGAAAAAGCAAAGAGCTTGGGGATTGACTATAT                            - TCCTTTGGAAGTGAGCCTCAAGGACACTGTGGAGAGCTTGAAGGAAA                             - AGAAGTTTCTGAAAGTT.                                                       ______________________________________                                    


3. The isolated nucleotide sequence according to claim 1, wherein saidsequence comprises the following cDNA sequence (SEQ ID NO.3):

    ______________________________________                                        GCAGAATCAACATAATCCACAATGAGCACCGCCCGCTGGAAAACTAGTC                             TGTGTCACCGGCGCTTCCGGTTACATCGCTTCCTGGCTCGTCAAGTTT                                 - CTTCTCGAACGCGGCTACACCGTGAAGGCCACCGTTCGCGACACGAA                             - TGATCCCAAAAAGGTAGATCACTTGCTCAGCCTTGATGGTGCGAAGGA                            - GAGATTGCATCTCGTGAAGGCGAATCTTCTAGAAGAAGGTTCCTTTGA                            - CTCTGCCGTTGAAGGTGTTCACGCTGTGTTTCACACTGCTTCTCCCTT                            - TTTCAACGATGCCAAAGATCCGCAGACTGAGTTGTTGGACCCGGCAGT                            - GAAGGGGACTCTGAATGTTCTGAAATCGTGTGTGAACTCGCCCACGCT                            - GAAACGCGTCGTTTTAACTTCTTCTATTGCTGCAGTTGCGTTCAGCGA                            - CAGGCCTAAAAACCCTGATGTTGTGGTTGACGAGACTTGGTATTCTGA                            - CCCGGAATACTGTAAGAGAACAGGGTTATGGTATAACCTTTCAAAGAC                            - TCTGGCCGAAGATGCTGCCTGGAAATTTGCGAAAGAAAACAACATTGA                            - CCTGGTTACAATGAACCCAGCATTGGTTGTTGTTGGACCTCTCTTGCAACC                         - AGTGCTTAATACTAGCGCTATAGTTTTAGGTTTAGTTAATGGGGC                               - GAAAACATTTAAAAATGCTTCTTTGGGATGGGTCGACGTGAAAGATGT                            - TGCATTGGCCCATATTCTAGCATATGAGAATGCTTCGGCTAATGGAAG                            - ATATTTACTAGTTGAGAGAGTAGCACACTTTGGAGATGCTGCCAAGAT                            - TTTACGAGATTTATATACCCAACATTGCAAATTCCAGACAAGTGTGAAGAC                         - GATAAGCCATTAGAACCAATATTTCAGGTTTCGAAGGAAAAAGCAAAG                            - AGCTTGGGGATTGACTATATTCCTTTGGAAGTGAGCCTCAAGGACACT                            - GTGGAGAGCTTGAAGGAAAAGAAGTTTCTGAAAGTTTAATGTTACCCT                            - TCAAGAAACGTGGAAACCTTCTTATGCCTATGCTTGAGGTAAACTTAGT                           - GTTAAATAAATGGAAAGTGATGTAAGTTCTGTAATAAGTGATTCTTGGA                           - CTGGTTTTTTCTGGTGACAAGTTTCAAAAATGAGTGACCATGGTAGAG                            - TCTGCGTTCCAGATTGTATCCTTGTGATATTGTATTAGTAGAAGATGTT                           - TGTTGCAGCTAATGCACCCATTATTATGTTTTTATCAAAAAAAAAAAAAA                          - A.                                                                       ______________________________________                                    


4. The isolated nucleotide sequence of claim 1 further comprising apromoter suitable for expression in prokaryotic or eukaryotic cells. 5.An isolated nucleotide sequence comprising the nucleotide sequence ofclaim 1, said nucleotide sequence further comprising a strong promoter.6. An isolated vector comprising the nucleotide sequence of claim
 1. 7.The isolated vector of claim 6, wherein said vector comprises binaryvector pGA.
 8. A recombinant prokaryotic or eukaryotic cell transformedwith the nucleotide sequence of claim
 1. 9. The recombinant prokaryoticor eukaryotic cell of claim 8, wherein said cell comprises a bacterialcell.
 10. The recombinant prokaryotic or eukaryotic cell of claim 9,wherein said cell is derived from E. coli or Agrobacterium tumefaciens.11. The recombinant cell of claim 8, wherein said cell is a plant cell.12. The recombinant cell of claim 11, wherein said plant cell is a plantseed cell.
 13. The recombinant cell of claim 11, wherein said cellcomprises a grapevine cell.
 14. A recombinant plant transformed with thenucleotide sequence of claim
 1. 15. A plant transformed with thenucleotide sequence of claim
 1. 16. The plant of claim 15, wherein saidplant is grapevine.
 17. Seed obtained from the plant of claim
 15. 18. Amethod for expressing a polypeptide compound with eutypine reductaseactivity in plants, comprising the steps of:a) transforming a plant cellwith the nucleotide sequence of claim 1; and b) regenerating of plantsfrom the transformed plant cell of step a) to obtain plants expressing aprotein with eutypine reductase activity.
 19. A method of treatingeutypa dieback in perennial ligneous plants comprising transforming saidplant with the isolated nucleotide sequence of claim
 1. 20. The methodof claim 19, wherein said plant is selected from the group consisting ofgrapevine, apricot, black currant, cherry, tamarind, almond, and apple.21. An isolated nucleotide sequence comprising a sequence which encodesa polypeptide capable of having eutypine reductase activity when saidpolypeptide is expressed in E. coli, and wherein said sequence is fromvigna radiata.
 22. An isolated nucleotide sequence comprising a sequencewhich encodes a polypeptide capable of having eutypine reductaseactivity when said polypeptide is expressed in E. coli, whichpolypeptide is recognized by antibodies raised against a polypeptide ofvigna radiata which has eutypine reductase activity, said polypeptide ofvignia radiata comprising the following amino acid sequence (SEQ IDNO:2):

    __________________________________________________________________________    Met Ser Thr Ala Ala Gly Lys Leu Val Cys Val Thr Gly Ala Ser Gly Tyr lle       Ala Ser Trp Leu Val Lys Phe Leu Leu Glu Arg Gly Tyr Thr Val Lys Ala Tyr         - Val Arg Asp Thr Asn Asp Pro Lys Lys Val Asp His Leu Leu Ser Leu Asp         - Gly Ala Lys Glu Arg Leu His Val Lys Ala Asn Leu Leu Glu Glu Gly Ser         - Phe Asp Ala Lys Asp Pro Gln Thr Glu Leu Asp Pro Val Lys                     - Phe Asn Ser Ala Val Glu Gly Val His Ala Val Phe His Thr Ala Ser Pro       Phe                                                                              - Gly Thr Leu Asn Val Leu Lys Ser Cys Val Asn Ser Pro Thr Leu Lys Arg      Val                                                                              - Val Leu Thr Ser Ser Ile Ala Ala Ala Val Ala Phe Ser Asp Arg Pro Lys      Asn Pro                                                                          - Asp Val Val Val Asp Glu Thr Tyr Tyr Ser Asp Pro Glu Tyr Cys Lys Arg      Thr                                                                              - Gly Leu Trp Tyr Asn Leu Ser Lys Thr Leu Ala Glu Asp Ala Ala Trp Lys      Phe                                                                              - Ala Lys Glu Asn Asn Ile Asp Leu Val Thr met Asn Ala Pro Ala Leu Val      Val Gly                                                                          - Pro Leu Leu Gln Pro Val Leu Asn Thr Ser Ala Ala Ile Val Leu Gly Leu      Val                                                                              - Asn Gly Ala Lys Thr Phe Lys Asn Ala Ser Leu Gly Trp Val Asp Val Lys      Asp                                                                              - Val Ala Leu Ala His Ile Leu Ala Tyr Glu Asn Ala Ser Ala Asn Gly Arg      Tyr                                                                              - Leu Leu Val Glu Arg Val Ala His Phe Gly Asp Ala Ala Lys Ile Leu Arg      Asp                                                                              - Leu Tyr Pro Thr Leu Gln Ile Pro Asp Lys Cys Glu Asp Asp Lys Pro Leu      Glu                                                                              - Pro Ile Phe Gln Val Ser Lys Glu Lys Ala Lys Ser Leu Gly Ile Asp Tyr      Ile Pro                                                                          - Leu Glu Val Ser Leu Lys Asp Thr Val Glu Ser Leu Lys Glu Lys Lys Phe      Leu                                                                              - Lys Val.                                                                 __________________________________________________________________________


23. A recombinant prokaryotic or eukaryotic cell transformed with thenucleotide sequence of claim
 21. 24. A recombinant prokaryotic oreukaryotic cell transformed with the nucleotide sequence of claim 22.25. A plant transformed with the nucleotide sequence of claim
 21. 26. Aplant transformed with the nucleotide sequence of claim
 22. 27. Theplant of claim 25, wherein said plant is grapevine.
 28. Seed obtainedfrom the plant of claim
 25. 29. A method for expressing a polypeptidecompound with eutypine reductase activity in plants, comprising thesteps of:(a) transforming a plant cell with the nucleotide sequence ofclaim 21; and (b) regenerating plants from the transformed plant cell ofstep (a) to obtain plants expressing a protein with eutypine reductaseactivity.
 30. The plant of claim 26, wherein said plant is grapevine.31. Seed obtained from the plant of claim
 26. 32. A method forexpressing a polypeptide compound with eutypine reductase activity inplants, comprising the steps of:(a) transforming a plant cell with thenucleotide sequence of claim 22; and (b) regenerating plants from thetransformed plant cell of step (a) to obtain plants expressing a proteinwith eutypine reductase activity.
 33. A plant transformed with thenucleotide sequence of claim 5.